The maternal metabolic environment can be detrimental to the health of the offspring. In a previous work, we showed that maternal high-fat (HH) feeding in rabbit induced sex-dependent metabolic adaptation in the fetus and led to metabolic syndrome in adult offspring. As early development representing a critical window of susceptibility, in the present work we aimed to explore the effects of the HH diet on the oocyte, preimplantation embryo and its microenvironment. In oocytes from females on HH diet, transcriptomic analysis revealed a weak modification in the content of transcripts mainly involved in meiosis and translational control. The effect of maternal HH diet on the embryonic microenvironment was investigated by identifying the metabolite composition of uterine and embryonic fluids collected in vivo by biomicroscopy. Metabolomic analysis revealed differences in the HH uterine fluid surrounding the embryo, with increased pyruvate concentration. Within the blastocoelic fluid, metabolomic profiles showed decreased glucose and alanine concentrations. In addition, the blastocyst transcriptome showed under-expression of genes and pathways involved in lipid, glucose and amino acid transport and metabolism, most pronounced in female embryos. This work demonstrates that the maternal HH diet disrupts the in vivo composition of the embryonic microenvironment, where the presence of nutrients is increased. In contrast to this nutrient-rich environment, the embryo presents a decrease in nutrient sensing and metabolism suggesting a potential protective process. In addition, this work identifies a very early sex-specific response to the maternal HH diet, from the blastocyst stage.

Heat stress can have severe deleterious effects on embryo development and survival. The present study evaluated whether CSF2 can protect the developmental competence of the bovine embryo following exposure to a heat shock of 41°C at the zygote and morula stages. In the first experiment, putative zygotes and 2-cell embryos were assigned to receive either 10 ng/ml CSF2 or vehicle, and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Heat shock reduced blastocyst development for embryos treated with vehicle but not for embryos cultured with CSF2. In the second experiment, day 5 embryos (morula) were treated with CSF2 or vehicle and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Temperature treatment did not affect development to the blastocyst stage and there was no effect of CSF2 treatment or the interaction. Results indicate that CSF2 can reduce the deleterious effects of heat shock at the zygote or two-cell stage when the embryo is transcriptionally inactive.

We report here the existence of bands of higher molecular weight after western blot analysis in three proteins – Skp1, p27 and IκBα in bovine preimplantation embryos. This finding is specific to preimplantation embryos (from the 2-cell stage to the blastocyst stage) and not differentiated fibroblast cells in which these bands were of expected molecular weight. We suggest that these bands of higher molecular weight represent a complex of proteins that are characteristic of preimplantation embryos.

The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1–10 μM for 6–24 h.

The Nlrp gene family contains 20 members and plays a pivotal role in the innate immune and reproductive systems in the mouse. During evolution, seven Nlrp4 gene copies (named from Nlrp4a to Nlrp4g). Nlrp4a–Nlrp4g have arisen that display specific or preferential ovarian expression patterns. However, the expression pattern and localization of Nlrp4g in mouse preimplantation embryo development are unknown. Here we report that Nlrp4g was highly expressed in mature oocytes and zygotes, then downregulated and not detected after the 2-cell embryo stage. NLRP4G protein remained present through the blastocyst stage and was mainly localized in the cytoplasm. Furthermore, overexpression of Nlrp4g in zygotes did not affect normal development in terms of the rate of blastocyst formation. These results provide the first evidence that NLRP4G is a maternal factor that may play essential role during zygotic genome activation in the mouse.

In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.

Cyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P<0.05). No statistical differences were found for blastocyst cell numbers. The mRNA expression for the EGR1 gene was down-regulated eight-fold in blastocysts that had been produced in vitro compared with their in vivo counterparts. Gene expression profiles for IGF2R, SLC2A8, COX2, DNMT3B and PCK2 did not differ among experimental groups. Bovine testis satellite I and Bos taurus alpha satellite methylation profiles from cAMP30aspiration protocol-derived blastocysts were similar to patterns that were observed in their in vivo equivalents (P > 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

The genome of all cells is protected at all times by mechanisms collectively known as DNA repair activity (DRA). Such activity is particularly important at the beginning of human life, i.e. at fertilization, immediately after and at the very onset of embryonic development. DRA in early development is, by definition, of maternal origin: the transcripts stored during maturation, need to control the integrity of chromatin, at least until the maternal/zygotic transition at the 4- to 8-cell stage in the human embryo. Tolerance towards DNA damage must be low during this critical stage of development. The majority of DNA damage is due to either apoptosis or reactive oxygen species (ROS). Apoptosis, abortive or not, is a common feature in human sperm, especially in oligoasthenospermic patients and FAS ligand has been reported on the surface of human spermatozoa. The susceptibility of human sperm to DNA damage is well documented, particularly the negative effect of ROS (Kodama et al., 1997; Lopes et al., 1998a, b) and DNA modifying agents (Zenzes et al., 1999; Badouard et al., 2007). DNA damage in sperm is one of the major causes of male infertility and is of much concern in relation to the paternal transmission of mutations and cancer (Zenzes, 2000; Aitken et al., 2003; Fernández-Gonzalez, 2008). It is now clear that DNA damaged spermatozoa are able to reach the fertilization site in vivo (Zenzes et al., 1999), fertilize oocytes and generate early embryos both in vivo and in vitro. The effect of ROS on human oocytes is not as easy to study or quantify. It is a common consensus that the maternal genome is relatively well protected while in the maturing follicle; however damage may occur during the long quiescent period before meiotic re-activation (Zenzes et al., 1998). In fact, during the final stages of follicular growth, the oocyte may be susceptible to damage by ROS. With regards to the embryo there is active protection against ROS in the surrounding environment i.e. in follicular and tubal fluid (El Mouatassim et al., 2000; Guerin et al., 2001). DNA repair activity in the zygote is mandatory in order to avoid mutation in the germ line (Derijck et al., 2008). In this review we focus on the expression of mRNAs that regulate DNA repair capacity in the human oocyte and the mechanisms that protect the embryo against de novo damage.

Preimplantation mammalian development culminates in the formation of a fluid-filled cavity, the blastocoele, which is a prerequisite for successful implantation and further development. The blastocoele is enclosed by a single layer of polarised cells, the trophectoderm, which is the first epithelium formed in development. In embryos of the mouse and the rabbit, a basolaterally located Na+/K+-ATPase hydrolyses ATP to drive the vectorial transport of ions, which is responsible for the accumulation of blastocoele fluid. Using non-invasive assays of energy substrate consumption and blastocoele fluid accumulation, experiments were carried out on single preimplantation rat embryos, to establish: (1) the roles of the Na+/K+-ATPase and exogenous energy substrates, and (2) the relationship between the consumption and metabolism of energy substrates and fluid accumulation, during blastocoele cavity formation in this species. Ouabain 0.5 mM and energy-substrate-free medium both caused an inhibition in the number of embryos forming a blastocoele in culture, and also reduced the rate of fluid accumulation by day 5 blastocysts collapsed in cytochalasin-D and allowed to re-expand. Ouabain also reduced the consumption of glucose (but not pyruvate) and the production of lactate by re-expanding blastocysts. In the absence of the inhibitor, a direct relationship was seen between fluid accumulation and both glucose (but not pyruvate) consumption and lactate production. However, ouabain had no effect on intact, expanded blastocysts. These results suggest that (1) a basolaterally located, ouabain-inhibitable Na+/K+-ATPase is involved in rat blastocoele formation, (2) this process is dependent on exogenous energy substrates, and (3) there may be a direct relationship between the metabolism of glucose via glycolysis, and blastocoele fluid accumulation.

DNA methylation is one of the proposed biochemical mechanisms involved in cell differentiation and in genomic imprinting, and DNA methyltransferase (DMT) is a key enzyme in the embryo since mutation of its gene is lethal early in development. In order to verify that non-viability of uniparental embryos was not due to a defect in the regulation of DMT activity, we compared the metabolism of methylation in parthenogenetic embryos (maternal genome) and in fertilised embryos (maternal and paternal genomes). As regards total methylation, estimated by a measure of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) formation, no significant difference was found between the two kinds of embryos during preimplantation development. Mean values were 4.5 ± 0.6 fmol (SAM +SAH)/h per 2-cell embryo and 0.40 ± 0.05 fmol SAH/h per 2-cell embryo, i.e. a SAH/(SAM + SAH) ratio of 9%; there was no detectable SAH formation in blastocysts. The same observation can be made for DMT activity, with mean values of: 7.8 fmol/h per oocyte, 8.5 fmol/h per 2-cell embryo, 6.1 fmol/h per 4-cell embryo, 4.1 fmol/h per morula, and no detectable activity in blastocysts. Total methylation as well as DNA methylation is characterised by a progressive drop in activity during preimplantation development.

Apoptosis may occur in early embryos in which the execution of essential developmental events has failed. Thus the initiation of the apoptotic mechanism may be related to activation of the embryonic genome. In this way, developmentally incompetent cells or whole embryos are eliminated. It is likely that some link exists between failed resumption of rRNA synthesis and the incidence of apoptosis in cleaving embryos. In this context, decreased developmental potential in cleaving nucleotransferred embryos is consistent with cell loss, and very likely due to programmed cell death. The effects of apoptosis inducers on cleaving embryos have not been characterised in comparable detail to that in the case of somatic cells. Early embryos provide a very good model for study of these processes because of the specificity of rRNA transcription resumption after fertilization. In our experiments three apoptosis inducers (staurosporin 10 mM, actinomycin D 0.05 mg/ml and camptothecin 0.1 mg/ml) were used in a culture medium for 15 h at the 4-cell stage (day 2) of mouse embryos, followed by further development in a pure culture medium until fixation on days 3, 4 and 5. In staurosporin-induced embryos, light microscopy immunostaining of nucleolar proteins (fibrillarin, Nopp140, protein B23) did not reveal changes in nucleolar morphology on day 3. On days 4 and 5, more compact (roundish) nucleoli (in comparison with controls) were observed. The embryos treated with camptothecin displayed a similar staining pattern to those with staurosporin at each day. In actinomycin-D-treated embryos, marked changes in nucleolar appearance were visible as early as day 3. These changes in nucleolar morphology consisted of loss of the reticulation appearance and fragmentation of nucleoli. In addition to nucleolar changes, significantly decreased cell proliferation was observed. The induced embryos did not reach the blastocyst stage. The number of blastomeres was decreased, and staining with Hoechst 33342 revealed a significant percentage of apoptotic nuclei (condensed/fragmented nuclei) from day 4.